In eukaryotic cells, DNA synthesis is restricted to a limited interval (S phase) in the cell division cycle. The DNA is functionally divided into many replicating units (replicons) which are duplicated at different but characteristic times within the S phase. Several lines of evidence indicate that protein synthesis is an obligate requirement for continuing DNA synthesis. In this study, we propose to establish a system in tissue culture cells in which we can ask the following question: are specific proteins elaborated at precise times before and during the S period which interact with specific nucleotide sequences in the eukaryotic genome in order to promote replication of these sequences at precise times? We require: 1) a cell line which has at least two marker nucleotide sequences - one which replicates early and the other late in the S period; each marker should be readily identifiable cytologically, and each should be separable from the rest of the genome by physical methods; 2) a method of synchrony in which all cells in the population enter and traverse S with a high degree of synchrony; with such a synchronized population, DNA can be labelled at precise times during S, and proteins can be prepared from the synchronized population at precise times; and 3) in vitro system in which semiconservative DNA replication occurs under optimum conditions, and which will initiate new chains in vitro upon the addition of appropriate macromolecules.